The induction of an antigen specific T cell response requires multiple interactions between cell surface receptors on a T cell and ligands on an antigen presenting cell. The primary interaction is between the T cell receptor/CD3 complex and a major histocompatibility complex molecule, which presents an antigenic peptide to the T cell receptor, thereby triggering an antigen specific signal in the T cells. In addition to this antigen specific signal, a T cell response require a second, costimulatory signal. A costimulatory signal can be generated in a T cell by stimulation of the T cell through a cell surface receptor CD28 (Harding, F. A. (1992) Nature 356:607-609). Ligands for CD28 have been identified on antigen presenting cells (APCs). CD28 ligands include members of the B7 family of proteins, such as B7-1 (CD80) and B7-2 (CD86) (Freedman, A. S. et al. (1987) J. Immunol. 137:3260-3267; Freeman, G. J. et al. (1989) J. Immunol. 143:2714-2722; Freeman, G. J. et al. (1991) J. Exp. Med. 174:625-631; Freeman, G. J. et al. (1993) Science 262:909-911; Azuma, M. et al. (1993) Nature 366:76-79; Freeman, G. J. et al. (1993) J. Exp. Med. 178:2185-2192). Additionally, B7-1 and B7-2 have been shown to bind another surface receptor on T cells related to CD28 termed CTLA4 (Linsley, P. S. (1991) J. Exp. Med. 174:561-569; Freeman, G. J. et al. (1993) Science 262:909-911). In contrast to CD28 which is constitutively expressed on T cells, CTLA4 is induced on T cells upon activation (Linsley, P. S. et al. (1992) J. Exp. Med. 176:1595-1604). Although a functional role for CTLA4 is unknown, there is some evidence that CTLA4 can synergize with CD28 in the delivery of a costimulatory signal to a T cell (Linsley, P. S. et al. (1992) J. Exp. Med. 176:1595-1604; Damle, N. K. et al. (1994) J. Immunol. 152:2686-2697).
Delivery of an antigen specific signal to a T cell in the absence of a costimulatory signal does not induce a T cell response, but rather has been found to induce a state of T cell unresponsiveness or anergy (see Schwartz, R. H. (1990) Science 248:1349; Jenkins, M. K. et al. (1988) J. Immunol. 140:3324). In a number of clinical situations it is desirable to inhibit T cell responses (e.g., in transplantation or autoimmune disorders). Thus, therapeutic approaches have been proposed to induce antigen specific T cell unresponsiveness by blocking of a costimulatory signal in T cells. For example, a CTLA4Ig fusion protein, which binds both B7-1 and B7-2, has been used to inhibit rejection of allogeneic and xenogeneic grafts (see e.g., Turka, L. A. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 11102-11105; Lenschow, D. J. et al. (1992) Science 257, 789-792). Similarly, antibodies reactive with B7-1 and/or B7-2 have been used to inhibit T cell proliferation and IL-2 production in vitro and inhibit primary immune responses to antigen in vivo (Hathcock K. S. et al. (1993) Science 262, 905-907; Azuma, M. et al. (1993) Nature 366:76-79; Powers, G. D. et al. (1994) Cell. Immunol. 153, 298-311; Chen C. et al. (1994) J. Immunol. 152, 2105-2114).
An alternative approach to anergy induction for avoiding an unwanted T cell response to an antigen is to clonally delete T cells specific for the antigen, thereby eliminating the antigen specific T cells from the T cell repertoire. In vivo, T cell maturation in the thymus involves clonal deletion of potentially autoreactive T cells. Additionally, there is increasing evidence that previously activated T cells are selectively depleted in the periphery after clonal expansion and effector function has occurred (Webb, S. et al. (1990) Cell 63:1249-1256; Rocha, B. et al. (1991) Science 251:1225-1227; and Russell, J. H. et al. (1991) Proc. Natl. Acad. Sci. USA 88:2151-2155). Deletion, or elimination, of many types of cells, including T cells, can occur by a mechanism termed apoptosis, or programmed cell death. The occurence of apoptosis in a cell is characterized by features including cell shrinkage, nuclear collapse and DNA fragmentation (reviewed in Cohen, J. J. et al. (1992) Ann. Rev. Immunol. 10:267-293). Several cell-surface molecules have been identified which, upon ligation, can induce apoptosis in a cell, including Fas and tumor necrosis factor receptors (Yonehara, S. et al. (1989) J. Exp. Med. 169:1747-1756; Trauth, B. C. et al. (1989) Science 245:301-305; Itoh, N. et al. (1991) Cell 66:233-239; and Greenblatt, M. S. et al. (1992) Blood 80:1339-1344;). However, none of these apoptotic molecules is restricted to the T cell lineage nor do they induce apoptosis in an antigen specific manner. The ability to clonally delete T cells in a manner dependent upon antigenic stimulation would provide a means for long-term inhibition of T cell responses in a variety of clinical situations without the need for chronic generalized immunosuppression of a subject with its attendant deleterious side effects.
This invention is based, at least in part, on the discovery of novel ligands which bind a T cell surface molecule and induce antigen specific apoptosis in an activated T cell. Preferably, the ligand is an antibody, or antibody fragment, which binds the T cell surface molecule CTLA4, in particular human CTLA4. Anti-CTLA4 antibodies which bind an epitope on CTLA4 that is distinct from the epitope(s) recognized by the known CTLA4 ligands B7-1 and B7-2 are particularly preferred for use in inducing antigen specific T cell apoptosis. The antibodies can be polyclonal or monoclonal antibodies, or fragments thereof. Chimeric and humanized monoclonal antibodies, and fragments thereof, are encompassed by the invention. In another embodiment, the ligand is a soluble recombinant form of a novel CTLA4 ligand distinct from B7-1 and B7-2 that can induce T cell apoptosis. Alternatively, the ligand can be a modified form of B7-1 or B7-2 that binds CTLA4 without binding CD28 (e.g., the modified form of B7-1 or B7-2 binds the same epitope on CTLA4 recognized by the antibodies of the invention and induces apoptosis in a T cell).
Another embodiment of the invention pertains to ligands which bind an epitope on CTLA4 which is distinct from an epitope bound by the known B7-1 and B7-2 ligands and which induces apoptosis in a T cell. Preferably, the CTLA4 epitope recognized by a ligand, e.g., antibody, of the invention includes or encompasses an amino acid sequence:
(Xaa)n-Leu-Thr-Phe-Leu-Asp-Asp-(Xaa)n (SEQ ID NO: 33),
wherein Xaa is any amino acids and n=0-20, preferably 0-5, more preferably 0-3. This CTLA4 epitope is found in human CTLA4 at amino acid positions 59 to 64. Thus, typically, Xaa are additional amino acid residues found at either the amino or carboxy side, or both the amino and carboxy sides, of the epitope in the human CTLA4 amino acid sequence (SEQ ID NO: 36). Alternatively, Xaa can be an amino acid residue which increases the solubility of the resulting peptide or enhances the immunogenicity of the resulting peptide for use as an immunogen. For example, Xaa can be a charged amino acid (e.g., lysine, arginine) which may be added to increase the solubility of the peptide. Alternatively, Xaa can be cysteines added to increase dimerization of the resulting peptide.
The ligands of the invention, when combined with a pharmaceutically acceptable carrier, can be used in compositions suitable for pharmaceutical administration.
The ligands of the invention are useful for clonally deleting activated T cells in an antigen specific manner, either in vitro or in vivo, by induction of T cell apoptosis. In one embodiment, an activated T cell is contacted in vitro with a first agent that stimulates the T cell through the TCR/CD3 complex and a second agent which crosslinks an epitope on CTLA4, or provides an intracellular signal through a CTLA4-mediated pathway, that induces apoptosis in the T cell. Alternatively, the second agent is administered to a subject, together with a pharmaceutically acceptable carrier, to induce T cell apoptosis in vivo in the subject. A preferred second agent is an anti-CTLA4 antibody of the invention. In addition to inducing apoptosis through a CTLA4-mediated pathway, one or more additional agents can be administered to a subject to inhibit delivery of stimulatory signals to T cells. For example, an agent which inhibits the production or function of a T cell growth factor(s) in the subject, such as an anti-IL-2 receptor antibody, an anti-IL-2 antibody or cyclosporin A, may also be administered to the subject. Alternatively or additionally, another agent which inhibits delivery of a costimulatory signal to T cells, such as a molecule (e.g., antibody or soluble fusion protein) which inhibits an interaction between CD28 and B7-1 and/or B7-2, may also be administered in conjunction with an agent which induces T cell apoptosis.
Clonal deletion of T cells by induction of apoptosis in accordance with the methods described herein is applicable to a variety of clinical situations. For example, alloreactive T cells can be deleted from a transplant recipient to inhibit rejection of transplanted cells, tissues or organs. Additionally, alloreactive T cells can be deleted from donor bone marrow prior to bone marrow transplantation to inhibit graft versus host disease in a transplant recipient. In other applications, autoreactive T cells are deleted to treat autoimmune disorders and allergen-specific T cells are deleted to treat allergies. Virally-infected or malignant T cells which express CTLA4 on their surface can also be eliminated according to the methods of the invention.
In addition to providing for induction of apoptosis in T cells, the invention also provides methods for inhibiting T cell apoptosis. In one embodiment, T cell apoptosis is inhibited by interfering with an interaction between CTLA4 on a T cell and a CTLA4 ligand that induces apoptosis on an antigen presenting cell. A blocking form of a CTLA4 antibody or fragment thereof (e.g., Fab fragment) or a blocking soluble form of the CTLA4 ligand can be used to inhibit T cell apoptosis. Alternatively, an agent which acts intracellularly to inhibit apoptosis in a T cell through a CTLA4-mediated pathway can be used. The methods for inhibiting apoptosis are useful for enhancing T cell responses, such as against tumor cells and pathogens (e.g., bacteria, viruses, fungi, parasites and the like) and for enhancing the efficacy of vaccination.